The overall goal of this project is to develop and validate new strategies for ameliorating tissue destruction of periodontal disease by modulating activity of immune effector cells and/or regulating expression of the cytokine, Receptor Activator of NF-?B Ligand (RANKL). Recent studies demonstrated unequivocally that host immune responses play a key role in activating osteoclasts and promoting bone resorption in periodontal disease. T and B lymphocytes found in periodontal lesions express abundant RANKL, and stimulation of RANKL expression in these lymphocytes in vitro induces osteoclastogenesis. These findings provide a fundamentally new understanding of the mechanism of periodontal disease and support the hypothesis that tissue destruction in periodontal disease can be ameliorated by inhibition of RANKL activity in the immune effector cells. This laboratory has developed, validated and published several inclusive experimental models to characterize the role of host immunity in periodontal disease. These models include: 1) three in vivo rat models to dissect the specific roles of T cells and B cells in mediating periodontal bone resorption in gingival challenge and adoptive transfer of T or B lymphocytes and an infection model of periodontal disease;and 2) two in vitro models for testing human peripheral blood lymphocytes from healthy persons or human gingival mononuclear cell osteoclastogenesis from patients with periodontal disease where these rats experience periodontal bone resorption. The objectives of this study are to determine whether inhibition of RANKL activity reduces periodontal bone resorption, and to characterize the cellular and immunological mechanisms that mediate this process. Aim 1 is to evaluate mechanisms of preventing or reducing periodontal bone resorption by inhibiting RANKL expression or modulating its activity In vitro and in vivo studies will characterize the effects of inhibiting RANKL directly by antibody and inhibiting the TNF-a-Converting Enzyme (TACE), which is required for RANKL solubilization, with anti-TACE antibody or the known TACE inhibitor, galardin. Outcome measures will include resorption of bone, as measured microscopically, and characterization of bone pit formation;Aim 2 is to evaluate mechanisms of preventing or reducing periodontal bone resorption by inhibiting signaling pathways that regulate RANKL production by lymphocytes. Studies will characterize the role of two key signaling molecules - TRAF6 and CDK4 - in regulating RANKL expression in activated rat T and B cells in vitro, and will evaluate the effects of blocking these signaling molecules in the three rat models. Aim 3 is to characterize the inhibitory mechanisms of periodontal bone resorption identified in Aims 1 and 2. in vitro studies will asses the effects of blocking RANKL or inhibiting TACE in healthy human peripheral blood T and B cells, and in gingival tissue mononuclear cells from patients with periodontal disease. Periodontitis, which is responsible for half of all tooth loss in adults, occurs in moderate form in 30% of American adults and in severe form in 5% of adults. The current standard of care is mechanical removal of plaque biofilms. Successful completion of these studies will provide a fundamental understanding of the role of host immune response in mediating bone resorption and may lead to future clinical translational studies of novel strategies for preventing tooth loss in periodontitis.